Antibodies serve important functions in basic and applied biomedical research, diagnostic tests for health care, and in cancer therapy. Many such applications require antibody engineering to improve the affinity and/or selectivity of the antibodies. Such antibody engineering studies will benefit from guidance provided by thorough site-directed mutagenesis experiments performed on a model system that is well characterized structurally. The purpose of the proposed studies is to understand the effect of a wide variety of site-directed mutations on the antigen-binding affinity and selectivity of an antibody that binds sequence-specifically to DNA. The proposed studies will also provide important information about molecular recognition in general and sequence-specific DNA recognition in particular. Inspection of the uncomplexed and antigen complexed structures of an anti- DNA antibody suggests a wide variety of site-directed mutations that will directly impact: Interdomain protein-protein interactions, the flexibility of an antigen binding loop, and electrostatic interactions with DNA. The specific aims of the proposed studies are to 1) Analyze existing experimental data for the purpose of understanding antibody sequence and structural data at these sites, 2) Perform detailed molecular modeling of the site-directed molecular modeling of the site-directed mutations, and 3) Make biochemical measurements of the antigen binding affinities and selectivities of the site-directed mutant proteins.